We appreciate the letter by Bosiljcic and colleagues raising questions about our studies published in Cancer Research, in which we showed that Gr-1+CD11b+ cells alter the premetastatic lung in an inflammatory and proliferative environment, diminish immune protection, and induce aberrant vasculature formation (1).

We understand and appreciate the concerns raised by the authors. We are aware that 4T1-GFP cells cause immune response as a result of the expression of the foreign protein GFP. However, the majority of the data were collected using 4T1 cells. The GFP-expressing 4T1 cells were used only in 2 experiments, in which the differentiation of tumor cells from other cells of the hosting organs was fundamentally important. This will be discussed in detail in the following text. We are sorry that we did not specify in our article wherein 4T1-GFP cells were used, and we apologize for the confusion caused by this. We would like to clarify here:

In Fig. 1, 4T1-GFP cells were used to differentiate tumor cells from other cells of the hosting organs. We tried to label 4T1 tumor cells with cell-tracking fluorescence dye, which has the problem of losing fluorescence over time. The 4T1-GFP provides better results than the current available approaches. We observed that 4T1-GFP cells were present in the lungs of 4T1-GFP tumor–bearing mice 14 days or later after tumor injection. The clear presence of these GFP-expressing cells in lungs in day 21 and many more in day 28 indicates that these cells were not rejected by the hosts. Because the purpose of the experiment was to establish the time line for the premetastatic phase, which is day 14 or earlier, we believe that the data provided in the Letter, showing that the anti-GFP IgG antibodies in the plasma of Balb/c mice implanted with 4T1GFP cells appeared only 3 week after tumor injection, suggest an unlikely strong immune response. Indeed, the strong GFP expressers often cause immune rejection and the weak expressers often induce immune tolerance and are able to survive. This is also observed by the authors as stated in the second paragraph of the Letter. Apparently different 4T1-GFP lines from different laboratories have shown such differences. Our 4T1-GFP cells express low levels of GFP and were not rejected under our experimental conditions.

4T1-GFP cells were injected through tail vein in the 4T1 tumor–bearing mice (14 days after tumor injection) to examine the blood vessel leakage in the premetastatic lung. Twenty-four hours later, the lungs were perfused with PBS and the lung sections were examined by GFP immunohistochemistry for the presence of 4T1-GFP cells. In this case, the short duration of 24 hours should likely cause no concern for immune rejection.

As mentioned earlier, the data related to the matrix metalloproteinase 9 in premetastatic lung are from 4T1 cells not expressing GFP. So there is no concern with the immune effect on the GFP-expressing 4T1 cells. We agree with the authors regarding the concerns about using 4T1-GFP tumor cells in Balb/c immunocompetent mice if without awareness, rational, and selection. In fact, many genetic mouse models expressing viral antigen likely have issues of host immune response; for example, MMTV-PyMT for breast tumors and K14-HPV16 for skin tumors (2, 3). Mouse models are not always perfect and have limitations. What needs to be done is to use these models wisely while addressing the problems and making improvements. One such improvement, for example, is transgenic mouse expressing low level of GFP.